2882 • The Journal of Neuroscience, April 1, 2020 • 40(14):2882–2894 Systems/Circuits Acetylcholine Modulates Cerebellar Granule Cell Spiking by Regulating the Balance of Synaptic Excitation and Inhibition X Taylor R. Fore, Benjamin N. Taylor, Nicolas Brunel, and XCourt Hull Department of Neurobiology, Duke University Medical School, Durham, North Carolina 27710 Sensorimotor integration in the cerebellum is essential for refining motor output, and the first stage of this processing occurs in the granule cell layer. Recent evidence suggests that granule cell layer synaptic integration can be contextually modified, although the circuit mechanisms that could mediate such modulation remain largely unknown. Here we investigate the role of ACh in regulating granule cell layer synaptic integration in male rats and mice of both sexes. We find that Golgi cells, interneurons that provide the sole source of inhibition to the granule cell layer, express both nicotinic and muscarinic cholinergic receptors. While acute ACh application can modestly depolarize some Golgi cells, the net effect of longer, optogenetically induced ACh release is to strongly hyperpolarize Golgi cells. Golgi cell hyperpolarization by ACh leads to a significant reduction in both tonic and evoked granule cell synaptic inhibition. ACh also reduces glutamate release from mossy fibers by acting on presynaptic muscarinic receptors. Surprisingly, despite these consistent effects on Golgi cells and mossy fibers, ACh can either increase or decrease the spike probability of granule cells as measured by noninvasive cell-attached recordings. By constructing an integrate-and-fire model of granule cell layer population activity, we find that the direction of spike rate modulation can be accounted for predominately by the initial balance of excitation and inhibition onto individual granule cells. Together, these experiments demonstrate that ACh can modulate population-level granule cell responses by altering the ratios of excitation and inhibition at the first stage of cerebellar processing. Key words: acetylcholine; cerebellum; EI balance; Golgi cell; granule cell; synaptic integration Significance Statement The cerebellum plays a key role in motor control and motor learning. While it is known that behavioral context can modify motor learning, the circuit basis of such modulation has remained unclear. Here we find that a key neuromodulator, ACh, can alter the balance of excitation and inhibition at the first stage of cerebellar processing. These results suggest that ACh could play a key role in altering cerebellar learning by modifying how sensorimotor input is represented at the input layer of the cerebellum. Introduction long been hypothesized to enable expansion recoding that en- To coordinate movements and learn sensorimotor associations, hances pattern separation and increases the encoding capacity of the cerebellum must integrate signals across a variety of modali- the network (Marr, 1969; Albus, 1971; Cayco-Gajic et al., 2017; ties and timescales (Mauk and Buonomano, 2004; Ito, 2006, Gilmer and Person, 2017). 2012; Giovannucci et al., 2017; Wagner et al., 2017). These signals While classical models of granule cell layer function posit are conveyed to the cerebellum via mossy fiber projections that rigid integration rules dictated by the anatomical pattern of form excitatory connections onto a much larger network of gran- mossy fiber to granule cell connectivity, recent studies have ule cells. The divergence from mossy fibers to granule cells has demonstrated that the granule cell layer can be contextually modified during behavior. Specifically, granule cell responses to certain sensory modalities can be suppressed during loco- Received Sept. 5, 2019; revised Feb. 3, 2020; accepted Feb. 20, 2020. motion (Ozden et al., 2012). Moreover, associative learning Authorcontributions:T.R.F.,B.N.T.,N.B.,andC.H.designedresearch;T.R.F.,B.N.T.,andC.H.performedresearch; can be enhanced during locomotion via circuit modulation T.R.F.,B.N.T.,andC.H.analyzeddata;T.R.F.,N.B.,andC.H.editedthepaper;T.R.F.,B.N.T.,andC.H.wrotethepaper. This work was supported by National Institutes of Health National Institute of Neurological Disorders and Stroke thought to occur in the granule cell layer (Albergaria et al., Grant5R01NS096289-02,NationalScienceFoundationGrantDGF1106401,theSloanFoundation,andtheWhitehall 2018). Such contextual modification of granule cell responses Foundation. We thank Dr. Lindsey Glickfeld and members of the C.H. and Glickfeld laboratories for input and may allow for both the enhancement of behaviorally relevant technical assistance throughout the project. stimuli that should be learned, as well as suppression of infor- The authors declare no competing financial interests. Correspondence should be addressed to Court Hull at [email protected]. mation that should not be learned. However, the cellular https://doi.org/10.1523/JNEUROSCI.2148-19.2020 mechanisms that enable such context-dependent regulation Copyright © 2020 the authors are largely unknown. Fore et al. • Cholinergic Modulation of Cerebellar Circuits J. Neurosci., April 1, 2020 • 40(14):2882–2894 • 2883 Throughout the brain, neuromodulators play a key role in Table 1. Convergence ratios and synaptic connection probabilities contextual circuit regulation (Letzkus et al., 2011; Froemke et al., Convergence Connection 2013; Kuchibhotla et al., 2017). In the cerebellum, there are N ratios probability prominent ACh projections that terminate in the granule cell mf 315 — — layer (Jaarsma et al., 1997). Moreover, immunohistochemical GrC 4096 — — studies have shown both nicotinic and muscarinic receptors in GoC 27 — — the cerebellar cortex (Neustadt et al., 1988; Dominguez del Toro mf ¡ GrC — 4:1 0.0127 et al., 1994; Nakayama et al., 1997; Dominguez de Toro et al., mf ¡ GoC — 10:1 0.0317 1997; Turner and Kellar, 2005). These anatomical observations GoC ¡ GrC — 4:1 0.1481 suggest that ACh could be a key regulator of cerebellar processing and cerebellar-dependent behaviors. Indeed, behavioral studies have demonstrated cholinergic enhancement of the optokinetic ings were performed at 32°C-33°C, digitized at 20 kHz (Digidata 1440A, reflex and vestibulo-ocular reflex (Tan and Collewijn, 1991, 1992; Molecular Devices), and filtered at 10 kHz. Glass monopolar electrodes Prestori et al., 2013). However, it remains unknown both how (1 M⍀) filled with aCSF, in conjunction with a stimulus isolation unit endogenous levels of cerebellar ACh are modulated in vivo and (ISO-Flex, A.M.P.I.), were used for extracellular stimulation of the mossy how ACh acts at the synaptic and circuit levels to modify cerebel- fiber tract. Cell-attached recordings were performed to avoid changes in lar cortical processing. granule cell excitability over time with whole-cell dialysis (Fleming and To test how ACh acts to modulate granule cell layer processing Hull, 2019). For these experiments, only cells with an initial spike prob- ability in control conditions between ϳ20% and 60% were recorded to and synaptic integration, we have investigated both cell-auto- allow for increases or decreases in spike rate during pharmacological nomous and circuit-level effects of ACh by recording from gran- manipulations. ule cell layer neurons in an acute, in vitro brain slice preparation. sIPCSs and evoked IPSCs were recorded at the EPSC reversal potential We find that ACh predominantly leads to a prolonged suppres- (10 mV). Evoked EPSCs were recorded at a holding potential of Ϫ70 mV sion of Golgi cell activity via muscarinic receptor activation, in and Ϫ60 mV for granule cells and Golgi cells, respectively. Voltage- turn reducing both tonic and evoked synaptic inhibition onto clamp recordings of IPSCs and EPSCs were collected using a cesium- granule cells. In addition, activation of presynaptic muscarinic based internal solution containing the following (in mM): 140 Cs- receptors on mossy fibers leads to a reduction in granule cell gluconate, 15 HEPES, 0.5 EGTA, 2 TEA-Cl, 2 MgATP, 0.3 NaGTP, 10 phosphocreatine-Tris , and 2 QX-314 Cl. pH was adjusted to 7.2 with excitation. Together, the coincident reduction in excitation 2 and inhibition increases spike probability in some granule cells, CsOH, resulting in a final osmolality of 310–315 mmol/kg. Current- clamp and voltage-clamp recordings of Golgi cells were performed using while reducing spike probability in others. A population-level a potassium-based internal solution containing the following (in mM): integrate-and-fire model of granule cell layer synaptic processing 150 K-gluconate, 3 KCl, 10 HEPES, 0.5 EGTA, 3 MgATP, 0.5 NaGTP, 5 reveals that the direction of modulation depends on the relative phosphocreatine-Tris2, and 5 phosphocreatine-Na2. pH was adjusted to balance of excitation and inhibition for individual granule cells. 7.2 with KOH (osmolality 315 mmol/kg). Membrane potentials were not Specifically, we find that the activity of granule cells with the most corrected for the liquid junction potential. Series resistance was moni- inhibition is preferentially enhanced by ACh, whereas the activity tored during voltage-clamp recordings witha5mVhyperpolarizing of granule cells with little inhibition is largely suppressed. Thus, pulse, and only recordings that remained stable over the period of data these data suggest that ACh can act to enhance the reliability of collection were used. All drugs were purchased from Abcam or Tocris granule cells that are significantly inhibited in response to specific Bioscience.